Emerging roles and mechanisms of miR-206 in human disorders: a comprehensive review

As a member of the miR-1 family, miR-206 is located between IL-17 and PKHD1 genes in human. This miRNA has been shown to be involved in the pathogenic processes in a variety of human disorders including cancers, amyotrophic lateral sclerosis, Alzheimer’s disease, atherosclerosis, bronchopulmonary dysplasia, coronary artery disease, chronic obstructive pulmonary disease, epilepsy, nonalcoholic fatty liver disease, Hirschsprung disease, muscular dystrophies, pulmonary arterial hypertension, sepsis and ulcerative colitis. In the current review, we summarize the role of miR-206 in both malignant and non-malignant situations and explain its possible therapeutic implications.


Introduction
MicroRNAs (miRNAs) are a group of naturally happening short non-coding RNAs with 21 to 22 nucleotide long. These transcripts contribute to post-transcriptional silencing of target genes [1,2]. A single miRNA can affect expression of thousands of mRNAs and their target genes [3,4]. Universally, miRNAs interact with 3′UTR to inhibit translation or degrade target transcripts. The critical seed region of miRNAs is located in the nucleotides 2-7 of their 5′UTR [5,6]. miRNAs participate in several critical regulatory functions associated with cell growth, developmental processes and differentiation. Dysregulation of miRNAs is associated with a wide array of human pathway pathologies, especially cancers [7,8]. Disruption in miRNA levels have been reported in numerous disease processes. So, they have the potential to be developed into novel therapeutic targets [9][10][11].
miR-206 is a member of the miR-1 family. The gene encoding this miRNA is located between the IL-17 and PKHD1 genes in human [5]. The cytogenetic band of miR-206 is 6p12.2. This miRNA has been shown to participate in the pathogenesis of a variety of malignant and non-malignant conditions. Like other member of this miRNA family, miR-206 has physiological roles as well. Members of mouse mir-1 family have important functions in muscle development [12]. In the current review, we summarize the role of miR-206 in both malignant and non-malignant conditions and explain its possible therapeutic implications.

Bioinformatics step
Prediction of miR-206 target genes miRWalk (http:// mirwa lk. umm. uni-heide lberg. de/), miRDB (http:// www. mirdb. org/), and TargetScan databases (https:// www. targe tscan. org/) were used to predict the miR-206 target genes. A total of 82 mRNAs have been identified as common targets of this miRNA in these three databases (Fig. 1). Based on the results, miR-206 is predicted to target a variety of genes being involved in a wide range of cellular functions.
remarkably enriched in the following GO terms: activation of cysteine-type endopeptidase activity involved in apoptotic process, collagen fibril organization, and hemopoiesis in biological process (BP), intracellular membrane-bounded organelle, actin cytoskeleton, and nucleus in cellular component (CC); and RNA polymerase binding, methyl-CpG binding, and DNA binding in molecular function (MF) ( Table 1).

Role of miR-206 in cancers
Several studies have assessed expression of miR-206 in different types of cancers and found the molecular mechanism of involvement of this miRNA in the carcinogenesis (Fig. 3).
An experiment in bladder cancer tissues has shown over-expression of lncRNA RMRP in these tissues compared with adjacent tissues using qRT-PCR method. As revealed by MTT assay and transwell assay, RMRP induces cell proliferation, migration and invasiveness of bladder cancer cells through regulation of miR-206. The latter finding is based on the observed binding of miR-206 and RMRP in luciferase assay [13].
Similarly, in breast cancer cells, miR-206 has a tumor suppressor role possibly through down-regulation of PFKFB3. Expression of miR-206 in estrogen receptor α (ERα) positive breast cancer cells has been found to be reduced by 17β-estradiol in a dose-dependent manner. Over-expression of miR-206 could impede production of fructose-2,6-bisphosphate, diminish lactate synthesis and reduce proliferative ability and migration of breast cancer cells [14]. An independent study in breast cancer has shown association between down-regulation of miR-206 and large tumor dimension and advanced clinical stage. Over-expression of miR-206 in MCF-7 cells has suppressed cell growth through hindering G1/S transition. This effect is mediated through suppression of expression of cyclin D2. Consistent with this finding, expression levels of miR-206 have been inversely correlated with those of cyclin D2 in breast cancer tissues [15]. Expression of miR-206 has also been shown to be reduced in ERα-positive breast tumors. Besides, expression of miR-206 has been inversely related with ERα but not ERβ transcript levels in breast cancer tissues. Forced over-expression of miR-206 into MCF-7 cells has led to reduction of cell growth in both dose-and time-dependent manners, implying that miR-206 can be a target for endocrine therapy in this type of cancer [16]. Similarly, miR-206 has been shown to inhibit stemness and metastastatic ability of breast cancer cells through influencing activity of MKL1/IL11 axis [17]. Moreover, this miRNA can suppress epithelial mesenchymal transition (EMT) through influencing activity of TGF-β signals in ER-positive breast cancer cells [18].           -miR-206 reduced proliferation and migration in TNBC patients [58] miR-206 has also been down-regulated in the cervical cancer tissues, parallel with up-regulation of its target gene c-Met as revealed by qRT-PCR assay and immunohistochemistry. Kaplan-Meier and log-rank analyses have shown relation between down-regulation of miR-206 and shorter overall survival. Besides, down-regulation of miR-206 in cervical cancer tissues has been associated with lymph node metastasis, advanced stage and advanced histological grade indicating the role of miR-206 in the metastasis and progression of cervical cancer. In fact, miR-206 has been found to be independent prognostic marker for overall survival of patients with this type of cancer [19]. Table 2 shows summary of the role of miR-206 in malignant conditions. Diagnostic role of miR-206 has been assessed in epithelial ovarian cancer [32], renal cell carcinoma [52] and rhabdomyosarcoma [53] (Table 3). In epithelial ovarian cancer, miR-206 levels can be used for discrimination of patients with incomplete response to platinum chemotherapy from those with complete response to this modality with area under the receiver characteristic curve (AUC) of 0.82 [32]. Most notably, expression levels of miR-206 has a high accuracy in discrimination of patients with rhabdomyosarcoma from healthy subjects with AUC value of 0.96 [53].

Role of miR-206 in non-malignant conditions
miR-206 has crucial effects in the pathophysiology of several non-malignant disorders through modulation of a variety of targets (Fig. 4).
Expression assays in amyotrophic lateral sclerosis (ALS) have shown dysregulation of circulatory levels of several miRNAs in these patients. Notably, miR-206 has been among up-regulated miRNAs in these patients. In addition, constant changes in miRNAs signature have been found to persist during progression of ALS. This finding indicates the potential of selected miRNAs such as miR-206 as longitudinal markers for this disorder [59].
Circulatory levels of a number of miRNAs such as miR-206 have also been used as possible predictors for the progression of amnestic mild cognitive impairment (aMCI) to Alzheimer's disease (AD). Notably, serum levels of miR-206 have been found to be higher in aMCI patients progressed to AD. Kaplan-Meier analysis has also demonstrated remarkable correlation between conversion of aMCI to AD and over-expression of miR-206 [60]. Over-expression of miR-206 in olfactory mucosal cells can also been used as an early diagnostic approach in AD [61]. Another experiment in animal models of AD and temporal cortex samples from AD patients has verified over-expression of miR-206. These effects are mainly mediated through modulation of BDNF expression. In fact, a neutralizing inhibitor of this miRNA could prevent the harmful effect of amyloid-β42 on BDNF and dendritic spine degeneration [62]. On the other hand, another study has shown a neuroprotective effect of miR-206-3p in AD [63]. Table 4 shows summary of the role of miR-206 in non-malignant conditions. Diagnostic value of miR-206 has been evaluated in AD and muscular dystrophies (Table 5). In Duchene muscular dystrophy, expression levels of miR-206 can be used as a diagnostic marker with AUC value of 0.96 [71]. Similarly, this miRNA can be used as a marker for diagnosis of Becker muscular dystrophy [77].

Discussion
miR-206 is an example of miRNAs with crucial roles in the pathogenesis of a wide range of human disorders. In the context of cancer, expression assays using qRT-PCR method and functional studies have led to the supposition of miR-206 as a tumor suppressor miRNA (summarized in Table 2), although some exceptions have been demonstrated [53]. It can reduce proliferation of cancer cells and induce their apoptosis [14] via different routes. Moreover, it can regulate cell cycle progression through modulation of expression of cell cycle-related genes [15]. The activity of several oncogenic pathways is modulated by miR-206. Examples of these pathways are EGF/EGFR [21], EGFR/MAPK [21], TGF-β [18], Notch3 [26], PTEN/ AKT/mTOR [29], VEGF [37], c-Met-Akt/Erk [42], Erk1/2 [42] and PI3K/AKT [43]. Most importantly, expression assays in patients with different responses to chemotherapeutic agents and functional studies in cell lines have shown that miR-206 can enhance cytotoxic effects of anti-cancer agents on cancer cells [22]. The latter finding highlights the importance of this miRNA in design of novel modalities to combat chemoresistance. Among non-malignant conditions, dysregulation of miR-206 has been reported in amyotrophic lateral sclerosis, Alzheimer's disease, atherosclerosis, bronchopulmonary dysplasia, coronary artery disease, chronic obstructive pulmonary disease, epilepsy, nonalcoholic fatty liver disease, Hirschsprung disease, muscular dystrophies, pulmonary arterial hypertension, sepsis and ulcerative colitis (summarized in Table 4). Thus, this miRNA can affect pathogenesis of a wide array of human disorders.
In silico studies have revealed that miR-206 can affect expression of tens of mRNAs being involved in the regulation of crucial cellular mechanisms such as activation of cysteine-type endopeptidase activity involved in apoptotic process, collagen fibril organization, hemopoiesis, regulation of tubulin deacetylation, regulation of vascular endothelial cell proliferation, regulation of gonadotropin secretion, response to prostaglandin, RNA polymerase binding, methyl-CpG binding and sequence-specific DNA binding. Therefore, it is not surprising that miR-206 influences pathoetiology of several disorders.
Notably, expression levels of miR-206 not only can be used for cancer diagnosis [53] and in determination of response to anti-cancer therapies [32], but also may be potential markers for discrimination of patients with muscular dystrophies from healthy subjects [71] or prediction of course of Alzheimer's disease [61]. Since miR-NAs can be easily tracked in the biofluids, these findings open a new era for detection of human disorders via noninvasive tools.

Conclusion
Altered expression of miR-206 in tumor tissues has been associated with malignant characteristics of cancers in terms of higher metastatic aptitude and lower survival rate, implying the role of this miRNA as a prognostic marker. Finally, forced over-expression of miR-206 in many cancer cell lines has led to reduction of malignant characteristics in cell line assays as well as animal models. Thus, this strategy can be used as a novel therapeutic approach for cancers. Meanwhile, miR-206 is involved in the pathophysiology of several non-malignant conditions, including neurodegenerative and neuropsychiatric     [81] disorders and muscular atrophies. Since efficient therapies for these kinds of disorders have not been developed yet, miR-206-targetted therapies might revolutionize this research field.